It is well known that plasma levels of apolipoprotein (apo)A-I and high density lipoprotein (HDL) are inversely correlated with the risk of cardiovascular disease (CVD). CVDs, which include heart attacks, stroke and high blood pressure, are estimated to shorten the average American life expectancy by about 10 years. Unfortunately, the mechanisms that protect the body from the pathological accumulation of lipid and cholesterol that cause CVD are not well understood. Recent studies have indicated that the ATP binding cassette transporter (ABCA1) is a critical cell surface protein required for the transfer of cellular lipid and cholesterol to lipid-free forms of apoA-I. The interaction of apoA-I and ABCA1 is key for the formation and maintenance of HDL levels in plasma and is likely important for the first step of reverse cholesterol transport from peripheral tissues including macrophages in the vessel wall. The long-term goal of this research is to understand this interaction in molecular detail. During the first funding period, we demonstrated that the C-terminal helical domain of apoA-I plays a critical role in interactions with the cell membrane under control of ABCA1. Our working model is that apoA-I interacts with ABCA1 through a two-step pathway that involves the tethering of apoA-I helix 10 to a cell surface lipid domain generated by the lipid translocase activity of ABCA1 followed by a direct protein-to-protein interaction between the two molecules. This scheme will be tested by a) determining the regions of apoA-I and ABCA1 that make contact during the protein-protein interaction, b) determining the characteristics of regions within other exchangeable apolipoproteins that are responsible for interaction with ABCA1, and c) identifying the nature of the cell surface lipid domain that is generated by ABCA1. A highly novel approach will be used that combines cutting edge mass spectrometry techniques with chemical cross-linking chemistry to derive new structural information on the apoA-I/ABCA1 interaction. This understanding will form a basis for new interventions designed to enhance apolipoprotein-mediated cholesterol efflux from cells, particularly those within the atherosclerotic vessel wall.